Dna insertional mutagenesis for the elucidation of a Photosystem II repair process in the green alga Chlamydomonas reinhardtii

The work outlines the isolation of transformant Chlamydomonas reinhardtii cells that appear to be unable to repair Photosystem II from photoinhibitory damage. A physiological and biochemical characterization of three mutants is presented. The results show differential stability for the D1 reaction center protein in the three mutants compared to the wild type and suggest lesions that affect different aspects of the Photosystem II repair mechanism. In the ag16.2 mutant, significantly greater amounts of D1 accumulate in the thylakoid membrane than in the wild type under steady-state growth conditions, and D1 loss is significantly retarded in the presence of the protein biosynthesis inhibitor chloramphenicol. Moreover, aberrant electrophoretic mobility of D1 in the ag16.2 suggests that this protein is modified to an as yet unknown configuration. These results indicate that the biosynthesis and/or degradation of D1 is altered in this strain. A different type of mutation occurred in the kn66.7 and kn27.4 mutants of C. reinhardtii. The stability of D1 declined much faster as a function of light intensity in these mutants than in the wild type. Thereby, the threshold of photoinhibition in these mutants was significantly lower than that in the wild type. It appears that kn66.7 and kn27.4 are similar conditional mutants, with the only difference between them being the amplitude of the chloroplast response to the mutation and the differential sensitivity they display to the level of irradiance.     

Isolation of cadmium sensitive mutants in Chlamydomonas reinhardtii by transformation/insertional mutagenesis

Abstract An arg7, cw15, mt⁺ strain of Chlamydomonas reinhardtii (CC1618) was transformed with pARG7.8, a plasmid containing the wild-type ARG7 gene. Over 2300 arg⁺ transformants were selected on TAP media. Upon subsequent analysis on TAP plus cadmium plates, five of the transformants failed to grow at a level of 400 μM cadmium and were designated as cadmium sensitive (Cd^s) mutants. Hybridization data indicated that vector (pBR329) sequences were present in these five mutants, but not in the untransformed parental strain. Two of the mutants have been back crossed to an arg7, cw15, Cd⁺, mt⁻ strain (CC425) and found to have progeny which always cosegregate the arg⁺ and Cd^s phenotypesin these two mutants results from the insertion of the plasmid pARG7.8 into a gene involving cadmium detoxification, and it provides a method by which to clone the interrupted gene(s).     

Comparative analysis of photosensitivity in photosystem I donor and acceptor side mutants of Chlamydomonas reinhardtii

Electron input from plastocyanin into photosystem I (PSI) is slowed down in the Chlamydomonas reinhardtii mutants affected at the donor side (PsaF or PsaB, lumenal loop j) of PSI. In contrast, electron exit from PSI to ferredoxin is diminished in the PSI acceptor side PsaC mutants K35E and FB₁. Although, the electron transfer reactions are diminished to a similar extent in both type of mutants, the PsaC mutants K35E and FB₁ are more light‐sensitive than the PsaF‐deficient strain 3bF or the PsaB mutants E613N and W627F. To assess the differential photosensitivity of donor and acceptor side mutants fluorescence transients, gross oxygen evolution and uptake, PSII photo‐inhibition and rate of recovery were measured as well as NADP⁺ photoreduction. The NADP⁺ photoreduction measurements indicated that the donor side is limiting the reduction rate. In contrast, measurements of gross oxygen evolution and uptake showed that the reducing side limits linear electron transfer. However, under high light, donor and acceptor side mutations lead to PSII photo‐inhibition and to a diminished rate of PSII recovery, cause lipid peroxidation and result in a decrease in the levels of PSI and PSII. The wild type is not affected under the same conditions. These responses are most pronounced in the PsaC‐K35E and PsaB‐W627F mutants, and they correlate with the light sensitivity of these strains. The correlation between limitation of electron transfer through PSI and the formation of reactive oxygen species as a cause for the light‐sensitivity is discussed.     

CrPP2C蛋白影响衣藻细胞鞭毛过渡区结构

目的:在莱茵衣藻细胞中构建并筛选鞭毛组装缺陷突变体,克隆缺陷基因,探索其对鞭毛组装的影响。方法:使用带有巴龙霉素(Paromomycin)抗性的基因片段随机插入衣藻细胞基因组中,通过性状筛选和基因序列分析获得与CrPP2C(Chlamydomonas reinhardtii type 2C protein phosphatase)基因相关的鞭毛异常突变体,根据突变体基本生物学性状和生化分析对CrPP2C基因的功能进行分析。结果:采用电转法成功获得衣藻细胞鞭毛缺陷相关突变体,部分细胞具有短鞭毛,部分细胞则不具有鞭毛;通过RESDA-PCR(restriction enzyme site-directed amplification PCR)对突变体基因序列分析,鞭毛缺陷性状由CrPP2C基因遭到破坏导致;把含有完整CrPP2C基因的重组质粒通过电转法导入突变体后,其鞭毛几乎恢复为野生型长度,并可检测到PP2C-HA融合蛋白的表达;观察鞭毛再生,突变体鞭毛只能再生为原有长度;使用药物处理使鞭毛缩短,突变体鞭毛能正常解聚;电镜检测突变体的鞭毛显微结构,发现过渡区的Y形结构缺陷。结论:CrPP2C基因的破坏导致鞭毛过渡区结构缺失,影响鞭毛组装过程,不组装鞭毛或组装短鞭毛。     

High light‐induced hydrogen peroxide production in Chlamydomonas reinhardtii is increased by high CO2 availability

The production of reactive oxygen species (ROS) is an unavoidable part of photosynthesis. Stress that accompanies high light levels and low CO₂ availability putatively includes enhanced ROS production in the so‐called Mehler reaction. Such conditions are thought to encourage O₂ to become an electron acceptor at photosystem I, producing the ROS superoxide anion radical () and hydrogen peroxide (H₂O₂). In contrast, here it is shown in Chlamydomonas reinhardtii that CO₂ depletion under high light levels lowered cellular H₂O₂ production, and that elevated CO₂ levels increased H₂O₂ production. Using various photosynthetic and mitochondrial mutants of C. reinhardtii, the chloroplast was identified as the main source of elevated H₂O₂ production under high CO₂ availability. High light levels under low CO₂ availability induced photoprotective mechanisms called non‐photochemical quenching, or NPQ, including state transitions (qT) and high energy state quenching (qE). The qE‐deficient mutant npq4 produced more H₂O₂ than wild‐type cells under high light levels, although less so under high CO₂ availability, whereas it demonstrated equal or greater enzymatic H₂O₂‐degrading capacity. The qT‐deficient mutant stt7‐9 produced the same H₂O₂ as wild‐type cells under high CO₂ availability. Physiological levels of H₂O₂ were able to hinder qT and the induction of state 2, providing an explanation for why under high light levels and high CO₂ availability wild‐type cells behaved like stt7‐9 cells stuck in state 1.     

Metabolism of hydrogen peroxide by the scavenging system in Chlamydomonas reinhardtii

The metabolism of hydrogen peroxide by the scavenging system was studied in Chlamydomonas grown in a selenium-lacking and a selenium-containing medium. In cells of the former, 40% of external hydrogen peroxide (H₂O₂) was scavenged by ascorbate peroxidase (AsAP; EC 1.11.1.11) and the residual H₂O₂ by catalase (EC 1.11.1.6). The enzymes involved in the ascorbate-glutathione cycle including AsAP. were localized in the chloroplast. In cells of the latter, glutathione peroxidase (GSHP; EC 1.11.1.9) functioned primarily in the removal of external H₂O₂. GSHP was located solely in the cytosol. The Chlamydomonas AsAP was relatively stable in ascorbate-depleted medium as compared with chloroplast AsAP of higher plants. No inactivation of the enzyme was found upon its incubation with hydroxyurea, an inhibitor of the chloroplast enzyme of higher plants. The enzyme showed higher specificity with pyrogallol than with ascorbate. The amino acid sequences in the N-terminal region of Chlamvdomonas AsAP showed no significant similarity to any other AsAP from higher plants and Euglena . The enzyme had a molecular mass of 34 kDa. The K_m values of the enzyme for ascorbate and H₂O₂ were 5.2±0.3 and 25±3.4 μ M , respectively. Hydrogen peroxide was generated at a rate of 6.1±0.8 μmol mg^-1 chlorophyll h^-1 in intact chloroplasts isolated from Chlamydomonas cells grown in the presence of Na-selenite, and it diffused from the organelles into the medium.     

Recovery of photosynthesis and phtosystem II fluorescence in Chlamydomonas reinhardtii after exposure to three levels of high light

Recovery from 60 min of photoinhibitory treatment at photosynthetic photon flux densities of 500, 1400 and 2200 μMmol m^?2 s^? was followed in cells of the green alga Chlamydomonas reinhardtii grown at 125 μMmol m^?2 s^?1. These light treatments represent photoregulation, moderate photoinhibition and strong photoinhibition, respectively. Treatment in photoregulatory light resulted in an increased maximal rate of oxygen evolution (P_max) and an increased quantum yield (Φ), but a 15% decrease in F_v/F_M. Treatment at moderately photoinhibitory light resulted in a 30% decrease in F_v/F_M and an approximately equal decrease in Φ. Recovery in dim light restored F_v/F_M within 15 and 45 min after high light treatment at 500 and 1400 μMmol m^?2 s^?1, respectively. Convexity (Θ), a measure of the extent of co-limitation between PS II turnover and whole-chain electron transport, and Φ approached, but did not reach the control level during recovery after exposure to 1400 μMmol m^?2 s^?1, whereas P_max increased above the control. Treatment at 2200 μMmol m^?2 s^?1 resulted in a strong reduction of the modeled parameters Φ, Θ and P_max. Subsequent recovery was initially rapid but the rate decreased, and a complete recovery was not reached within 120 min. Based on the results, it is hypothesized that exposure to high light results in two phenomena. The first, expressed at all three light intensities, involves redistribution within the different aspects of PS II heterogeneity rather than a photoinhibitory destruction of PS II reaction centers. The second, most strongly expressed at 2200 μmol m^?2 s^?1, is a physical damage to PS II shown as an almost total loss of PS II_α and PS II Q_B-reducing centers. Thus recovery displayed two phase, the first was rapid and the only visible phase in algae exposed to 500 and 1400 μmol m^?2 s^?1. The second phase was slow and visible only in the later part of recovery in cells exposed to 2200 μmol m^?2 s^?1.     

Identification of a gene for UDP-sulfoquinovose synthase of a green alga, Chlamydomonas reinhardtii, and its phylogeny.

Sulfoquinovosyl diacylglycerol is responsible for the structural and functional integrity of the photosystem II complex of a green alga, Chlamydomonas reinhardtii. We cloned a cDNA of C. reinhardtii containing an open reading frame for a protein 36-64% identical in the primary structure to known UDP-sulfoquinovose synthases, which are required for SQDG synthesis, in other organisms. Through the introduction of the cDNA, a cyanobacterial disruptant as to the UDP-sulfoquinovose synthase gene recovered the ability to synthesize sulfoquinovosyl diacylglycerol, thus confirming that the cDNA encodes the UDP-sulfoquinovose synthase. On the genome, the cDNA was divided into 14 exons, and the gene designated as SQD1 was present as one copy. The molecular phylogenetic tree for the UDP-sulfoquinovose synthase showed grouping of C. reinhardtii together with species that require sulfoquinovosyl diacylglycerol for the functioning of the PSII complex, but not with those that do not utilize the lipid for photosynthesis. The role of sulfoquinovosyl diacylglycerol in the functioning of the photosynthetic membranes might evolve in harmony with the system of the membrane lipid synthesis such as UDP-sulfoquinovose synthase gene.     

Effect of dichromate on photosystem II activity in xanthophyll-deficient mutants of Chlamydomonas reinhardtii

The photosystem II activity and energy dissipation was investigated when algal Chlamydomonas reinhardtii genotypes were exposed to dichromate toxicity effect. The exposure during 24 h to dichromate effect of two C. reinhardtii mutants having non-functional xanthophylls cycle, as npq1 zeaxanthin deficient and npq2 zeaxanthin accumulating, induced inhibition of PSII electron transport. After dichromate-induced toxicity, PSII functions of C. reinhardtii mutants were investigated under different light intensities. To determine dichromate toxicity and light intensity effect on PSII functional properties we investigated the change of energy dissipation via PSII electron transport, non-photochemical regulated and non-regulated energy dissipation according to Kramer et al. (Photosynth Res 79:209–218, 2004). We showed the dependency between dichromate toxicity and light-induced photoinhibition in algae deficient in xanthophyll cycle. When algal mutants missing xanthophylls cycle were exposed to dichromate toxicity and to high light intensity energy dissipation via non-regulated mechanism takes the most important pathway reaching the value of 80%. Therefore, the mutants npq1 and npq2 having non-functional xanthophylls cycle were more sensitive to dichromate toxic effects.     

Isolation and characterisation of chemotactic mutants of Chlamydomonas reinhardtii obtained by insertional mutagenesis

The swimming behaviour of the green flagellated protist Chlamydomonas reinhardtii is influenced by several different external stimuli including light and chemical attractants. Common components are involved in both the photo- and chemo-sensory transduction pathways, although the nature and organisation of these pathways are poorly understood. To learn more about the mechanism of chemotaxis in Chlamydomonas, we have generated nonchemotactic strains by insertional mutagenesis. The arginine-requiring strain arg7-8 was transformed with DNA carrying the wild-type ARG7 gene. Of the 8630 arginine-independenttransformants obtained, five are defective in their chemotaxis towards various sugars. Two of the mutants (CTX2 and CTX3) are blocked only in their response to xylose. Mutant CTX1 is blocked in its response to xylose, maltose and mannitol, but displays normal taxis to sucrose. Mutants CTX4 and CTX5 lack chemotactic responses to all sugars tested. CTX1, CTX4 and CTX5 represent novel chemotactic phenotypes not previously obtained using ultra-violet or chemical mutagenesis. Genetic analysis confirms that each mutation maps to a single nuclear locus that is unlinked to the mating-type locus. Further analysis of CTX4 indicates that the mutant allele is tagged by the transforming ARG7 DNA. CTX4 appears to be defective in a component specific for chemotactic signal transduction since it exhibits wild-type photobehavioural responses (phototaxis and photoshock) as well as the wild-type responses of EGTA-induced trans-flagellum inactivation and acid-induced deflagellation. Insertional mutagenesis has thus permitted the generation of novel chemotactic mutants that will be of value in the molecular dissection of the signalling machinery.     

Mutation of Residue Arginine~(18)of Cytochrome b_(559)a-Subunit and its Effects on Photosystem II Activities in Chlamydomonas reinhardtii

It has been known that arginine is used as the basic amino acid in the a-subunit of cytochrome b_(559)(Cyt b_(559)) excepthistidine. However, previous studies have focused on the function of histidine in the activities of photosystem (PS) II andthere are no reports regarding the structural and/or functional roles of arginine in PSII complexes. In the present study,two arginine (R18) mutants of Chlamydomonas reinhardtii were constructed using site-directed mutagenesis, in whichR18 was replaced by glutamic acid (E) and glycine (G). The results show that the oxygen evolution of the PSII complexin the R18G and R18E mutants was approximately 60% of wild-type (WT) levels and that, after irradiation at high lightintensity, oxygen evolution for the PSII of mutants was reduced to zero compared with 40% in WT cells. The efficiency oflight capture by PSII (F_v/F_m) of R18G and R18E mutants was approximately 42%-46% that of WT cells. Furthermore, levelsof the a-subunit of Cyt b_(559) and PsbO proteins were reduced in thylakoid membranes compared with WT. Overall, thesedata suggest that R18 plays a significant role in helping Cyt b_559 maintain the structure of the PSII complex and its activity,although it is not directly bound to the heme group.     

Structure‐based design of novel Chlamydomonas reinhardtii D1‐D2 photosynthetic proteins for herbicide monitoring

The D1‐D2 heterodimer in the reaction center core of phototrophs binds the redox plastoquinone cofactors, Q_A and Q_B, the terminal acceptors of the photosynthetic electron transfer chain in the photosystem II (PSII). This complex is the target of the herbicide atrazine, an environmental pollutant competitive inhibitor of Q_B binding, and consequently it represents an excellent biomediator to develop biosensors for pollutant monitoring in ecosystems. In this context, we have undertaken a study of the Chlamydomonas reinhardtii D1‐D2 proteins aimed at designing site directed mutants with increased affinity for atrazine. The three‐dimensional structure of the D1 and D2 proteins from C. reinhardtii has been homology modeled using the crystal structure of the highly homologous Thermosynechococcus elongatus proteins as templates. Mutants of D1 and D2 were then generated in silico and the atrazine binding affinity of the mutant proteins has been calculated to predict mutations able to increase PSII affinity for atrazine. The computational approach has been validated through comparison with available experimental data and production and characterization of one of the predicted mutants. The latter analyses indicated an increase of one order of magnitude of the mutant sensitivity and affinity for atrazine as compared to the control strain. Finally, D1‐D2 heterodimer mutants were designed and selected which, according to our model, increase atrazine binding affinity by up to 20 kcal/mol, representing useful starting points for the development of high affinity biosensors for atrazine.     

Identification and regulation of high light-induced genes in Chlamydomonas reinhardtii

We have used restriction fragment differential display for isolating genes of the unicellular green alga Chlamydomonas reinhardtii that exhibit elevated expression on exposure of cells to high light. Some of the high light-activated genes were also controlled by CO2 concentration. Genes requiring both elevated light and low CO2 levels for activation encoded both novel polypeptides and those that function in concentrating inorganic carbon (extracellular carbonic anhydrase, low CO2-induced protein, ABC transporter of the MRP subfamily). All the genes in this category were shown to be under the control of Cia5, a protein that regulates the responses of C. reinhardtii to low-CO2 conditions. Genes specifically activated by high light, even under high-CO2 conditions, encoded a 30 kDa chloroplast membrane protein, a serine hydroxymethyltransferase, a nuclease, and two proteins of unknown function. Experiments using DCMU, an inhibitor of photosynthetic electron transport, and mutants devoid of either photosystem I or photosystem II activity, showed aberrant expression of all the genes regulated by both CO2 and high light, suggesting that redox plays a role in controlling their expression. In contrast, there was little effect of DCMU or lesions that block photosynthetic electron transport on the activity of genes that were specifically controlled by high light.     

Mutation of Residue Arginine^18 of Cytochrome b559 α-Subunit and its Effects on Photosystem Ⅱ Activities in Chlamydomonas reinhardtii

It has been known that arginine is used as the basic amino acid in the α-subunit of cytochrome bsss （Cyt bsss） except histidine. However, previous studies have focused on the function of histidine in the activities of photosystem （PS） Ⅱ and there are no reports regarding the structural and/or functional roles of arginine in PSll complexes. In the present study, two arginine18 （R18） mutants of Chlamydomonas reinhardtii were constructed using site-directed mutagenesis, in which R18 was replaced by glutamic acid （E） and glycine （G）. The results show that the oxygen evolution of the PSII complex in the R18G and R18E mutants was approximately 60% of wild-type （WT） levels and that, after irradiation at high light intensity, oxygen evolution for the PSll of mutants was reduced to zero compared with 40% in WT cells. The efficiency of light capture by PSll （Fv/Fm） of R18G and R18E mutants was approximately 42%-46% that of WT cells. Furthermore, levels of the α-subunit of Cyt bsss and PsbO proteins were reduced in thylakoid membranes compared with WT. Overall, these data suggest that R18 plays a significant role in helping Cyt bss9 maintain the structure of the PSll complex and its activity, although it is not directly bound to the heme group.     

Polyphasic rise of chlorophyll a fluorescence in herbicide-resistant D1 mutants of Chlamydomonas reinardtii

Chlorophyll (Chl) a fluorescence transient, a sensitive and non-invasive probe of the kinetics and heterogeneity of the filling up of the electron acceptor pool of Photosystem II (PS II), was used to characterize D1-mutants of Chlamydomonas reinhardtii. Using a shutter-less system (Plant Efficiency Analyzer, Hansatech, UK), which provides the first measured data point at 10 s and allows data accumulation over several orders of magnitude of time, we have characterized, for the first time, complete Chl a fluorescence transients of wild type (WT), cell wall less (CW-15) C. reinhardtii and several herbicide-resistant mutants of the D1 proteins: D1-V219I A251V, F255Y, S264A G256D and L275F. In all cases, the Chl a fluorescence induction transients follow a pattern of O-J-I-P where J and I appear as two steps between the minimum Fo (O) and the maximum Fmax (Fm, P) levels. The differences among the mutants are in the kinetics of the filling up of the electron acceptor pool of PS II (this paper) in addition to those in the re-oxidation kinetics of Q_A ^– to Q_A, published elsewhere (Govindjee et al. (1992) Biochim Biophys. Acta: 1101: 353–358; Strasser et al. (1992) Archs. Sci. Genève 42: 207–224) and not in the ratio of the maximal fluorescence Fm to the initial fluorescence Fo. The value of this experimental ratio is Fm/Fo = 4.4±0.21 independent of the mutation. At 600 W m^–2 of 650 nm excitation, distinct hierarchy in the fraction of variable Chl a fluorescence at the J level is observed: S264A > A251V G256A > L275F V219I > F255Y CW-15 WT. At 300 and 60 W m^–2 excitation, a somewhat similar hierarchy among the mutants was observed for the intermediate levels J and I. Addition of bicarbonate-reversible inhibitor formate did not change the O to J phases, slowed the I to P rise, and in many cases, slowed the decay of fluorescence beyond the P level. These observations are interpreted in terms of formate effect being on the acceptor rather than on the donor side (S-states) of PS II. The formate effect was different in different mutants, with L275F being the most insensitive mutant followed by others (V219I, F255Y, WT, A251V and S264A). Further, in the presence of high concentrations of DCMU, identical transients were observed for all the mutants and the WT.The quantum yield of photochemistry of PS II, calculated from 1-(Fo/Fm), is in the range of 0.73 to 0.82 for the WT as well as for the mutants examined. Thus, in contrast to differences in the kinetics of the electron acceptor side of PS II, there were no significant differences in the maximum quantum yield of PS II, among the mutants tested. We suggest that earlier photochemistry yield values were much lower (0.4–0.6) than those reported here due to either higher measured values of Fo by instruments using camera shutters, or due to the use of cells grown in less than-optimal conditions.

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Both spillover and light absorption cross-section changes are involved in the regulation of excitation energy distribution between the two photosystems during state transitions in wheat leaf

Weak red light-induced changes in chlorophyll fluorescence parameters and in the distribution of PS I and PS II in thylakoid membranes were measured in wheat leaves to investigate effective ways to alter the excitation energy distribution between the two photosystems during state transition in vivo. Both the chlorophyll fluorescence parameter Fm/Fo and F685/F735, the ratio of fluorescence yields of the two photosystems at low temperature (77 K), decreased when wheat leaves were illuminated by weak red light of 640 nm, however, Fm/Fo decreased to its minimum in a shorter time than F685/F735. When Photosystem (PS II) thylakoid (BBY) membranes from adequately dark-adapted leaves (control) and from red light-illuminated leaves were subjected to SDS-polyacrylamide gel electrophoresis under mildly denaturing conditions, PS I was almost absent in the control, but was present in the membranes from the leaves preilluminated with the weak red light. In consonance with this result, the content of Cu, measured by means of the energy dispersive X-ray microanalysis (EDX), increased in the central region, but decreased in the margin of the grana stacks from the leaves preilluminated by the red light as compared with the control. It is therefore suggested that: (i) both spillover and absorption cross-section changes are effective ways to alter the excitation energy distribution between the two photosystems during state transitions in vivo, and the change in spillover has a quicker response to the unbalanced light absorption of the two photosystems than the change in light absorption cross-section, and (ii) the migration of PS I towards the central region of grana stack during the transition to state 2 leads to the enhancement of excitation energy spillover from PS II to PS I.     

The herbicide-resistant D1 mutant L275F of Chlamydomonas reinhardtii fails to show the bicarbonate-reversible formate effect on chlorophyll a fluorescence transients

Herbicide-resistant mutants of the eukaryotic green alga Chlamydomonas reinhardtii, that are altered in specific amino acids in their D-1 protein, show differential bicarbonate-reversible formate effects. These results suggest the involvement of D1 protein in the bicarbonate effect. A 25 mM formate treatment of mixotrophically or photoautotrophically grown wild type cells results in a slower rise of chlorophyll a fluorescence transient followed by a dramatically slowed decline during measurements in continuous light. These effects are fully reversed upon addition of 10 mM bicarbonate. The mutant BR-202 [L275F] is, however, highly insensitive to 25 mM formate suggesting that a significant change in formate (bicarbonate) binding has occurred in helix V of the D1 protein near histidine involved in Fe binding. With the exception of DCMU-4 [S264A], which is considerably more sensitive to formate than the wild type, five other different [V219I, A25IV, F255Y, G256D and cell-wall deficient CW-15] mutants display a relatively similar response to formate as wild type. Absence of formate effect on a PS II-lacking [FuD-7] mutant confirms the sole involvement of PS II in the bicarbonate effect.     

Protein tyrosine phosphorylation in the transition to light state 2 of chloroplast thylakoids

Redox dependent protein phosphorylation in chloroplast thylakoids regulates distribution of excitation energy between the two photosystems of photosynthesis, PS I and PS II. Several thylakoid phosphoproteins are known to be phosphorylated on N-terminal threonine residues exposed to the chloroplast stroma. Phosphorylation of light harvesting complex II (LHC II) on Thr-6 is thought to account for redistribution of light energy from PS II to PS I during the transition to light state 2. Here, we present evidence that a protein tyrosine kinase activity is required for the transition to light state 2. With an immunological approach using antibodies directed specifically towards either phospho-tyrosine or phospho-threonine, we observed that LHC II became phosphorylated on both tyrosine and threonine residues. The specific protein tyrosine kinase inhibitor genistein, at concentrations causing no direct effect on threonine kinase activity, was found to prevent tyrosine phosphorylation of LHC II, the transition to light state 2, and associated threonine phosphorylation of LHC II. Possible reasons for an involvement of tyrosine phosphorylation in light state transitions are proposed and discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Studies of excitation energy transfer within the green alga Chlamydomonas reinhardtii and its mutants at 77 K

The 77 K picosecond fluorescence of intact Chlamydomonas reinhardtii exhibits a 680-nm band (F680) that can be identified with light-harvesting chlorophyll. Analysis of the time and spectral dependence of F680 reveal a forward transfer rate of 1/(15 ps) from this 680-nm species to photosystem II. The possibility of transfer through LHC I, the light-harvesting complex closely associated with photosystem I with a transfer time of 60 to 100 ps, is indicated by analysis of similar data in the 700–720 nm region. Simple kinetic models that account for the time dependence of the emissions F707, F703 and F715 are proposed.Based in part on a thesis submitted in partial fulfillment of the requirements for the Ph.D. Degree, University of Rochester (SL).     

Time-resolved chlorophyll fluorescence studies on photosynthetic mutants of Chlamydomonas reinhardtii: origin of the kinetic decay components

The room temperature chlorophyll fluorescence decay kinetics of photosynthetic mutants of Chlamydomonas reinhardtii have been measured as a function of Photosystem 2 (PS2) trap closure, DNB-induced quenching at F_M, and time-resolved emission spectra. The overall decays have been analyzed in terms of three or four kinetic components where necessary. A comparison of the characteristics of the decay components exhibited by the mutants with the wild-type has been carried out to elucidate the precise origins of the different emissions in relation to the observed pigment-protein complexes. It is shown that a) charge recombination in PS2 is not necessary for the presence of long-lived decay components, b) there are two rapid PS1-associated emissions (=30 and 150–200 ps), c) a slow PS1 decay is observed (=1.73 ns) in the absence of PS1 reaction centres, d) the two variable components (=0.25–1.2 and 0.5–2.2 ns) observed in the wild-type arise from LHC2 and e) a rapid (=50–250 ps) decay is associated with the PS2 core antenna (CP3 and CP4). These results show that the intact thylakoid membrane system is too complex to distinguish all of the individual kinetic components.Abbreviations Aexp preexponential factor (Amplitude) - chl chlorophyll - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl urea - DNB m, dinitrobenzene - F_M maximum chl fluorescence level - F₀ initial chl fluorescence level - F_v variable chl fluorescence (F_M–F₀) - LHC light harvesting chl a/b protein complex - PS photosystem - Q_A primary stable electron acceptor of PS2